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J Theor Biol. Author manuscript; available in PMC 2018 March 07.
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Published in final edited form as:

J Theor Biol. 2017 March 07; 416: 28–37. doi:10.1016/j.jtbi.2016.12.021.
Mathematical Modeling of the Methionine Cycle and

Transsulfuration Pathway in Individuals with Autism Spectrum
Disorder
Troy Vargasona, Daniel P. Howsmonb, Stepan Melnykc, S. Jill Jamesc, and Juergen Hahna,b,*
aDepartmentof Biomedical Engineering, Rensselaer Polytechnic Institute, 110 8th Street, Troy,
NY 12180 USA
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bDepartment of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, 110 8th
Street, Troy, NY 12180 USA
cDepartment of Pediatrics, University of Arkansas for Medical Sciences, 4301 West Markham
Street, Little Rock, AR 72205 USA
Abstract
Previous research has shown a connection between metabolic abnormalities in the methionine
cycle and transsulfuration pathway and autism spectrum disorder. Using clinical data from a case-
control study investigating measurements of transmethylation and transsulfuration metabolites, a
steady-state model of these metabolites in liver cells was developed and participant-specific
parameters were identified. Comparison of mean parameter values and parameter distributions
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between neurotypical study participants and those on the autism spectrum revealed significant
differences for four model parameters. Sensitivity analysis identified the parameter describing the
rate of glutamylcysteine synthesis, the rate-limiting step in glutathione production, to be
particularly important in determining steady-state metabolite concentrations. These results may
provide insight into key reactions to target for potential intervention strategies relating to autism
spectrum disorder.
Keywords
Personalized model; transmethylation; steady-state analysis; parameter estimation; sensitivity
analysis
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*
Corresponding author. hahnj@rpi.edu.
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Conflicts of Interest
The authors declare that they have no conflict of interest.
Author Contributions
TV participated in the design of the study, performed the analysis, interpreted results, and drafted the manuscript; DPH participated in
the design of the study, and helped to interpret results and draft the manuscript; SM and SJJ developed the methodology for collection
of the clinical data, and collected and interpreted this data; JH conceived of the study and participated in its design and coordination,
and helped to interpret results and draft the manuscript. All authors read and approved the final manuscript.
Vargason et al. Page 2
1. Introduction
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Autism spectrum disorder (ASD) is a general diagnosis for a group of neurodevelopmental

disabilities that appear in the early years of childhood, typically before the age of two
(American Psychiatric Association, 2013). Although the disorder can be associated with a
large array of symptoms, the two primary criteria for diagnosis of ASD are difficulties with
social interaction and communication and the display of restricted, repetitive behaviors
(American Psychiatric Association, 2013). The Center for Disease Control’s most recent
estimate of ASD prevalence among children in the United States is 1 in 68 (Christensen et
al., 2016). This is a substantial increase from its 1996 estimate of 1 in 294 (Yeargin-Allsopp
et al., 2003), and even more so from international estimates in the early 1970s of
approximately 1 in 2300 (Gillberg and Wing, 1999).
The significant rise in ASD prevalence has prompted research into certain risk factors for the
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disorder. While there is clearly a genetic component involved in the development of ASD
(Abrahams and Geschwind, 2008; Bailey et al., 1995; Rai, 2016), recent twin studies
indicate that heritability of the disorder is potentially lower than previously estimated
(Gaugler et al., 2014; Hallmayer et al., 2011). Environmental factors are also suggested to
contribute to increased ASD susceptibility through a variety of mechanisms (Rossignol et
al., 2014). For example, a recent study found significant correlations between the
concentrations of organic pollutants, such as pesticides, in the blood of children and the
severity of ASD-associated behaviors in those children with the disorder potentially also
affected by a child’s genetic susceptibilities (Boggess et al., 2016). The results of another
study indicated a significant association between maternal antidepressant treatment before
pregnancy and ASD risk in children (Castro et al., 2016). Correlations have also been found
between concentrations of certain toxic metals in blood and urine and ASD severity (Adams
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et al., 2012; Adams et al., 2016). While there is an ongoing debate on what factors
contribute to ASD (and in what capacity), these findings provide evidence that the factors
involved in ASD risk are much more complex than just genetic predisposition alone.
Recent research has also pointed to a critical connection between incidence of ASD and
irregularities in folate-dependent one-carbon metabolism and transsulfuration. Several
studies have found evidence for reduced methylation capacity and increased oxidative stress
in people with ASD compared to age-matched controls (Adams et al., 2011; James et al.,
2006; Melnyk et al., 2012). Since the metabolic pathways directly responsible for these
abnormalities are the methionine cycle and transsulfuration pathway, these results suggest
that some dysfunction in these pathways might be associated with ASD. While the
methionine cycle is found in all cells in the body, transsulfuration is limited to the liver,
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pancreas, small intestine, kidney, and brain (Finkelstein and Martin, 2000; Vitvitsky et al.,
2006). Combined, these pathways have many diverse functions in the human body, including
the regulation of gene expression through addition of methyl groups to DNA (Ulrey et al.,
2005), myelin protein stabilization in nerve cells (Miller, 2003), and synthesis of
glutathione, one of the body’s major antioxidants (Wu et al., 2004). Glutathione plays an
important role in detoxification and removal of reactive oxygen species in the body. In
mammals, glutathione is found in all tissue types, with high concentrations found in the liver
(Lu, 1999) where it is primarily synthesized. The main function of glutathione is antioxidant

defense against reactive oxygen species, including free radicals (Fang et al., 2002), and
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detoxification of environmental toxins, including heavy metals (Adams et al., 2011).

Metabolic abnormalities and deficiency of glutathione can result in increased intracellular
oxidative stress. Studies suggest that elevated levels of oxidative stress are associated with
the pathophysiology of a number of diseases, including Alzheimer’s disease (Markesbery,
1997), diabetes (Giugliano et al., 1996), and cystic fibrosis (Roum et al., 1993), as well as
ASD (James et al., 2006). Although the link between glutathione, oxidative stress, and
disease has been well-studied, exact explanations for why these relationships exist have yet
to be found (Ballatori et al., 2009).
This paper seeks to contribute to our understanding of how metabolites of the methionine
cycle and transsulfuration pathway interact by creating a mathematical model where the
probability density functions (PDFs) of the parameter values are determined from clinical
data. Detailed models of the methionine cycle and transsulfuration pathway exist (Duncan et
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al., 2013a, 2013b; Reed et al., 2008; Reed et al., 2004) that include complex nonlinear
formulas for the rates of each of the reactions, as well as inhibitory and excitatory effects of
metabolites on enzymes. These models have a large number of parameters which cannot all
be estimated by measuring only a small number of metabolites in the blood. Instead, we
chose to develop a smaller model of the pathways with unidirectional linear kinetics. This
model does not have the biological detail of the larger models, but it has the advantage of
having only 8 parameters (the kinetic rate constants), which can be determined from clinical
data. Estimation of the PDFs of these parameters for both neurotypical study participants
and participants with ASD, as well as performing sensitivity analysis on the model, allows
for identification of important reactions that could potentially be manipulated for future
intervention strategies for ASD.
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2. Materials and Methods

2.1. Plasma metabolite data
The clinical data used in this model come from the Integrated Metabolic and Genomic
Endeavor (IMAGE) study at Arkansas Children’s Hospital Research Institute (Melnyk et al.,
2012). The IMAGE study protocol was approved by the University of Arkansas for Medical
Sciences’ Institutional Review Board, and parents provided written informed consent. Data
for 82 neurotypical study participants (control) and 93 participants on the autism spectrum
(case) were used for the model in this work. These data reflect plasma concentrations of
transmethylation and transsulfuration metabolites, as well as those of tyrosine, nitrotyrosine,
and chlorotyrosine, quantified with high performance liquid chromatography (HPLC)
(Melnyk et al., 1999; Melnyk et al., 2000). Amounts of cytosine, 5-methylcytosine, and 8-
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oxo-deoxyguanosine in DNA were also quantified using a HPLC-ultraviolet system and

HPLC electrochemical detection (Helbock et al., 1998). The interested reader is referred to
(Melnyk et al., 2012) for further information regarding data collection and experimental
procedures.

2.2. Model development

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The components and structure of the methionine cycle and transsulfuration pathway can be
found in the public literature (Reed et al., 2008). The mathematical model developed here is
based upon the structure of these metabolic pathways in liver cells, where 8 metabolites
measured in the clinical study are modeled. The model consists of the component balances
of the individual metabolites at steady state, is based on mass action kinetics, and can be
mathematically described as a set of linear algebraic equations. Molar concentrations of
metabolites (c), participant-specific rate parameters (p), and efflux rate constants (f)
represent the inputs to the system.
A compartmental model, where the concentrations of the metabolites within each

compartment are considered to be well-mixed, is used. There are potentially fluxes of
components into and out of each compartment, resulting in a net flux rate. Similarly,
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reactions within each compartment can result in the concentration of a metabolite being
increased or decreased. This is represented by the general component balance:
(1)
The structure of the metabolic reactions described by the mathematical model is shown in
Figure 1. Methionine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and
homocysteine make up the methionine cycle. Cysteine, glutamylcysteine, glutathione
(GSH), and glutathione disulfide (GSSG) are the modeled components of the
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transsulfuration pathway. Cystathionine, an intermediate in the reaction of homocysteine

converting to cysteine, was omitted from the transsulfuration pathway due to lack of
available measurements for it.
The model consists of 8 equations (Table 1) and contains 9 participant-specific parameters

represented by variables p1 through p9, each having units of hr−1. Included are also 3 global
efflux rate constants representing the removal rates of SAM, homocysteine, and GSH from
the compartment. These flux rates are represented by variables f2 = 9 hr−1 (Duncan et al.,
2013a; Stabler and Allen, 2004), f4 = 0.001 hr−1 (Chwatko and Jakubowski, 2005; Duncan
et al., 2013a; Refsum et al., 1985), and f7 = 3.8 hr−1 (Hong et al., 2005). To maintain the
steady state properties of the model, a zero-order influx rate constant u is used to supply
methionine to the system. This variable has units of μM/hr and is equal to the sum of a
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participant’s three efflux rates to achieve a steady state balance:
(2)
with f2, f4, and f7 being the removal rates (in hr−1) of SAM, homocysteine, and GSH,
respectively, and c2, c4, and c7 being the concentrations (in μM) of SAM, homocysteine, and
GSH, respectively.

In this model, methionine enters the system at the rate defined in Equation 2, and is
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converted into SAM with rate constant p1. SAM is then converted to SAH in a DNA
methylation reaction with rate constant p2, while some of it is also lost via breakdown or
excretion (represented by efflux rate constant f2). SAH turns into homocysteine according to
the rate constant p3. Homocysteine then follows several paths: it is re-methylated to form
methionine with rate constant p4, contributes to the transsulfuration pathway by converting
to cysteine with rate constant p5, and leaves the system with rate constant f4. Cysteine is
converted to glutamylcysteine according to rate constant p6, with glutamylcysteine
subsequently being depleted to form GSH with rate constant p7. GSH is transported out of
liver cells or degraded (represented by flux rate constant f7) or is oxidized to GSSG at a rate
defined by p8. GSSG is reduced back to GSH according to rate constant p9.
Each participant-specific rate parameter in the model represents the activity of an enzyme
associated with the methionine cycle or transsulfuration pathway. For example, p1
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corresponds to the combined activities of methionine adenosyltransferase I (MAT-I) and

methionine adenosyltransferase III (MAT-III), two enzymes responsible for the conversion
of methionine to SAM. p2 describes the combined rate of the enzymes DNA
methyltransferase (DNMT), glycine N-methyltransferase (GNMT), and other
methyltransferases, while p3 characterizes the activity of S-adenosylhomocysteine hydrolase
(SAHH) and p4 captures the combined effects of methionine synthase (MS) and betaine-
homocysteine methyltransferase (BHMT). p5 describes a lumped reaction of homocysteine
being converted directly to cysteine with no cystathionine intermediate; this parameter is
taken to represent solely the activity of cystathionine β-synthase (CBS), which is responsible
for the conversion of homocysteine to cystathionine. Parameter p6 corresponds to glutamate-
cysteine ligase (GCL), while p7 describes the activity of glutathione synthetase (GS). p8
captures the rate of glutathione peroxidase (GPX), and p9 describes the action of glutathione
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reductase (GR). These model enzymes are summarized in Table 2.
2.3. Parameter estimation

When estimating parameters for each study participant, the values of c and f as given in
Table 1 are known; these are the measured metabolite concentrations and defined efflux
rates, respectively. The values of p represent the unknown participant-specific rate
parameters and need to be computed. Since the relationship between GSH and GSSG is
underdetermined in this system, however, the individual values of p8 and p9 cannot be
computed; instead, only the ratio of p8 to p9 (expressed p8:p9 or GPX:GR) can be calculated.
This ratio was thus considered to be one combined term describing the relative values of p8
and p9. To account for this, the component balance equation for GSSG (Table 1) was
rewritten as:
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(3)
This effectively reduces the model to 8 parameters. Given that the model is still linear, it can
be written such that the c values are in an 8×8 coefficient matrix multiplied by an 8×1 vector
of unknown p variables. This product is then summed with an 8×1 vector of the f·c efflux

terms, and this sum is set equal to the 8×1 zero vector. Formulating the model in this manner
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allows for estimation of the 8 unknown p values by solving a system of linear equations.
Estimation of model parameters was performed for each study participant in the
neurotypical and ASD groups using their individual measured values of c from the clinical
data.
2.4. Model assumptions

Certain assumptions needed to be made for this work to develop the mathematical structure
of the model and explain how it is affected by the availability of data:
1. The derived model has a linear structure and includes 8 parameters to allow for
the distributions of the parameter values to be estimated from the clinical data.
While a more detailed model of the pathway may allow for a more accurate
description, it is assumed that a model of the chosen complexity can reflect the
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pathway behavior reasonably well.
2. The model system is at steady state, so the concentrations of metabolites are not
changing over time. This has to be assumed because each study participant only
had metabolite measurements taken at one point in time. Along with this, it is
assumed that the combined contributions of an enzyme’s concentration and
activity do not change over time.
3. Changes in concentrations of metabolites in plasma are reflected by proportional

changes in concentrations of metabolites in liver cells, although this is not always
true in some cases (Duncan et al., 2013a). However, this assumption must be
made here because the clinical data consist of measurements taken in plasma
while the model structure describes the metabolic pathways in liver cells.
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Other assumptions were made to explain the biological implications of each reaction in the
model and how these were also affected by the available data:
4 The conversion of methionine to SAM is due to the combined effects of MAT-I

and MAT-III. Having only a single measurement in time for each metabolite
does not allow for understanding the effects of individual enzymes acting in
parallel, so only the combined effect is modeled.
5 The conversion of SAM to SAH is due to the combined contributions of DNMT

enzymes, GNMT, and approximately 150 other methyltransferases. For reasons
given in Assumption 4, only the combined effect of these enzymes is modeled.
Additionally, GNMT uses glycine as a second substrate, but the associated
reaction is treated as pseudo first order so only the effects of SAM concentration
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are considered.
6 While the reaction converting SAH to homocysteine by the enzyme SAHH is

reversible, it is represented by one net forward rate constant in the model. At
steady state, it is not possible to determine the individual rate constants of the
forward and reverse reactions. However, the rate of the forward reaction must be
greater than the rate of the reverse reaction for the system to remain at steady
state.

7 The conversion of homocysteine to methionine is due to the combined

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contributions of MS and BHMT. This assumption is made for reasons similar to

Assumption 4, so again only the combined contribution of these enzymes is
modeled. Additionally, BHMT uses betaine as a second substrate, but the
associated reaction is treated as pseudo first order so only the effects of
homocysteine concentration are considered.
8 The reactions involving the conversion of homocysteine to cystathionine and the

conversion of cystathionine to cysteine are represented by a single lumped
reaction where homocysteine is directly converted to cysteine. This is due to no
measurements being taken for cystathionine.
Furthermore, an assumption was made to focus the analyses on just the model system of
interest:
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9 There are no indirect inhibitory or regulatory effects between metabolites in the

described pathways. In addition, it is assumed that there are no interaction
effects from other metabolic pathways. While it is known that other pathways
and metabolites, such as folate, have long-range regulatory effects (Reed et al.,
2008), these mechanisms are ignored in this model so that parameters can be
estimated as stated in Section 2.3.
The final assumption was needed to reflect that certain information is not readily available
for subsets of participants:
10 The rates of SAM removal, homocysteine excretion, and GSH degradation are
identical in neurotypical and ASD study participants. No reported numbers for
the values of these rates are available for participants on the autism spectrum.
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Therefore, the reported values of these rates for neurotypical individuals were
assumed to be identical to those for participants on the autism spectrum.
2.5. Sensitivity analysis

The sensitivity of a model to a specific parameter is commonly measured as the change in
the model’s output due to a perturbation of that parameter (Frey and Patil, 2002). Local
sensitivity analysis can be performed by individually perturbing each parameter from its
baseline value while keeping the values of all other parameters constant. This is also referred
to as a one-factor-at-a-time approach (Hamby, 1994; Saltelli and Annoni, 2010). A simple
method for perturbing a chosen parameter is to add or subtract a percentage of its base value
(Hamby, 1994).
The baseline value of each model parameter was taken to be the mean value of that
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parameter among all neurotypical participants. The resulting set of baseline parameters,
which were then known, corresponded to a particular unknown set of metabolite
concentrations, which were treated as the outputs of the system. However, calculating the
output’s sensitivity to changes in parameters required knowledge of the baseline
concentrations that would serve as a point of reference for the model output. This required
calculating the metabolite concentrations that corresponded to the baseline parameter values.

To solve for these unknown baseline concentrations, an approach similar to that described in
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Section 2.3 was taken. Using the model equations in Table 1, an 8×8 matrix of average p and
f terms was constructed and multiplied by an 8×1 vector of unknown baseline c values.
Their product was then set equal to an 8×1 zero vector to form a homogeneous system of
linear equations. Given that any row of the matrix is equal to the negative sum of all other
rows, all rows are not linearly independent; the calculated rank of the matrix was then found
to be 7. Since the largest potential rank of this matrix is 8, the rank-nullity theorem states
there exists one solution for the right null space of this matrix. The right null space was then
solved for by using the null command in MATLAB. The absolute values of these vector
elements were taken to be the baseline metabolite concentrations for the purposes of
sensitivity analysis.
Each model parameter, as well as each efflux rate constant, was perturbed according to the
one-factor-at-a-time approach. To observe the output’s response to small perturbations,
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parameters were increased and decreased by 1% of their baseline value. The metabolite
concentrations corresponding to each new perturbation were then recalculated by finding the
right null space of the parameter matrix as described above. Each new metabolite
concentration resulting from a given perturbation was compared to its baseline value using
the relative change, which was used to account for large differences in magnitude between
metabolite concentrations. The sensitivity measure for a particular perturbation was taken to
be the Euclidean norm of the vector containing these 8 relative changes (one for each
metabolite). Total sensitivity of the output to changes in a certain parameter was calculated
as the average of this sensitivity measure for the negative and positive perturbations of that
parameter.
2.5.1 Sensitivity analysis and cross validation—The many model assumptions and
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the uncertainties in model parameter estimates may limit the confidence with which the
results of sensitivity analysis can be interpreted. Thus, these results were validated using a
leave-one-out cross validation approach (Kohavi, 1995). The cross-validation procedure was
applied by removing the first neurotypical participant’s set of parameters from the data – this
participant was treated as the test set, while the remaining 81 participants were treated as the
training set. Sensitivity analysis was performed, as described above, independently for the
training set and the test set. The obtained sensitivity measures were recorded, the first
participant’s data were replaced, and the process was repeated one at a time for each of the
other neurotypical participants. Thus, sensitivity analysis was performed for 82 training sets
and 82 test sets. The averages of the sensitivity measures between the training sets and test
sets were then compared. Performing the analysis in this manner helped to ensure that the
sensitivity results were robust to changes in the available sample set. Efflux rates were also
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considered in this cross-validation.
3. Results
3.1. Parameter estimation
The 8 participant-specific parameters were estimated for the 82 neurotypical participants and
93 participants with ASD for who concentrations of all 8 model metabolites were available

in the clinical data. Means and standard deviations for metabolite concentrations among the
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participants included in this study are provided in Table 3. Significant statistical differences
were observed between the neurotypical and ASD groups for all metabolites except
homocysteine (α = 0.01).
Following the methodology outlined in Section 2.3, a system of linear equations was
constructed using a coefficient matrix of c values, a vector of unknown p values, and a
vector of efflux terms f·c. However, the matrix of c values was found to be rank deficient
(for reasons identical to those given in Section 2.5 for a matrix of p and f terms). The four
components describing the methionine cycle were specifically responsible for this condition.
Finding a unique solution for the associated parameters required that a constraint be placed
on one of the four parameters in the methionine cycle. To determine the optimal parameter
to constrain, an equality constraint was applied to one parameter in the cycle, and values of
the remaining parameters were estimated. The constraint was then removed and placed on
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another parameter in the methionine cycle, and the new group of unconstrained parameters
was estimated. This process was repeated for all four parameters in the methionine cycle.
Estimates of all model parameters resulting from each of the constraints for neurotypical
participants were compared with approximated biological values of these rate constants from
the literature. The lsqlin routine in MATLAB was used to define each constraint and
estimate the parameters such that the constraints were satisfied.
The equality constraint used to estimate parameters relates the rate of each reaction in the
methionine cycle to a nominal rate, given in μM/hr. Each constraint has the form:
(4)
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where pi is a participant’s estimated value of the i-th rate constant (to be solved), ci is that
participant’s concentration of the i-th metabolite, pnom is a nominal value for the rate
parameter being estimated (taken from the literature), and is the average concentration of
the i-th metabolite for the group that the participant belongs to (neurotypical or ASD). Each
constraint is only applied to a reaction within the methionine cycle, i.e., i ranges from 1 to 4.
During the process of determining the optimal constraint, only data for neurotypical
participants were considered. When the constraint described by Equation 4 was applied to
each methionine cycle parameter in turn, the parameter estimates in Table 4 were obtained.
Approximate nominal values from the literature are also provided. The linear structure of the
transsulfuration pathway results in the estimates for parameters p5 through p8:p9 remaining
unchanged across the different constraints. Placing a constraint on p1 produces very large
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values for the other methionine cycle parameters. Constraining p2 gives much smaller
estimates of p1 and p3, with unacceptable negative values for p4. The constraint on p3,
however, produces parameter estimates that are most similar to the values from the literature.
Values for p2 and p3 resulting from the constraint on p4 are too large to consider acceptable.
Thus, constraining p3 is the preferred approach to estimating parameters for the methionine
cycle.

Using the optimal constraint on p3, parameter values for ASD participants were then
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estimated. A comparison of the estimates for control and ASD participants is provided in
Table 5. Significant statistical differences can be seen between the neurotypical and ASD
groups for all parameters except p3 and p7 (α = 0.01). Additional insight into these
differences can be gained from observing the respective PDFs of each parameter for the
control and ASD groups. Using kernel density estimation with a normal kernel (using the
ksdensity command in MATLAB), PDFs were constructed for each parameter (Figure 2).
The most apparent differences between neurotypical and ASD distributions are seen for
parameters p1 (Figure 2A), p2 (Figure 2B), p4 (Figure 2D), and p8:p9 (Figure 2H). Although
p5 and p6 (Figure 2E and 2F, respectively) displayed significant statistical differences, there
are not observable differences in the distributions for the two groups of participants. The two
distributions for p3 (Figure 2C) are nearly identical, which is a consequence of the constraint
placed upon the parameter.
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3.3. Sensitivity analysis

Metabolite concentrations were considered to be the outputs of the system for local
sensitivity analysis. Since the system is linear, local sensitivity analysis results for positive
and negative 1% perturbations should be identical. However, both perturbations were
performed for each parameter to ensure the results were not affected by numerical
inaccuracies. The results of both perturbations were indeed found to be almost identical, and
the average results are reported in Table 6.
The metabolite concentrations were found to be most sensitive to changes in parameter p6,
which describes the rate at which cysteine is converted to glutamylcysteine. In other words,
altering the value of p6 causes the largest cumulative relative change in the steady state
metabolite concentrations. It is thus an important parameter and has the most influence on
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the model output. After p6, the output was most sensitive to changes in parameter p5, which
defines the rate of production of cysteine from homocysteine, and to changes in efflux rate
constant f7, which represents the removal rate of GSH from the system. The model output
also showed relatively high sensitivity to changes in parameter p4, which describes the rate
at which homocysteine is converted to methionine. These results indicate that these
parameters also have a greater effect on the model concentrations. The remaining
participant-specific parameters, having equally low measures of sensitivity, were indicated to
have less effect on steady state metabolite concentrations. Efflux rate constants f2 and f4
appeared to have minimal influence on the concentrations of metabolites. Additionally, the
cross-validation results (Table 6) revealed no effective differences between the parameter
sensitivity measures as calculated from all samples, from the averaged training results, and
from the averaged test results.
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4. Discussion
This work developed a model of the methionine cycle and transsulfuration pathway and this
is the first effort which estimates parameters for such a model using clinical data from both
neurotypical and ASD study participants. Furthermore, the distributions of the parameters

were estimated and compared between the groups of study participants, as each participant
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had different metabolite concentrations, resulting in different model parameters.
Development of the presented model was based on a number of assumptions. Some of these
assumptions may limit the accuracy of the model, while others may have little or no bearing
on the model’s ability to describe true metabolic behavior. The assumption of a steady state
system can be particularly limiting since biological systems are rarely, if ever, in this state of
equilibrium. That being said, few physiological measurements, especially if they involve
metabolites in clinical studies, are taken over time and as such this is a reasonable
assumption for this particular application. Additionally, assuming linear interactions
between enzymes and substrates may neglect to capture the true behavior of these reactions.
Michaelis-Menten equations are often used to describe the nonlinear kinetics of enzymes,
but the kinetic parameters of these equations are difficult to estimate without available time-
series data. In general, the assumptions made are based upon the state-of-the-art of what can
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be done in a clinical research trial. Regardless of the implications of these assumptions, the
overall purpose of this study was to characterize and model metabolic differences between
neurotypical and ASD participants. The current model provides a starting point for
describing the interactions of the studied metabolic pathways in ASD.
The most significant limitation of the model is that the efflux rate constants are not
necessarily specific to each participant. Given the variability in metabolite concentrations
between participants, it is unlikely that these efflux rates are identical for participants within
the same group, let alone identical between the neurotypical and ASD participant groups.
Differences in these efflux rates could explain the differences observed for certain
participant-specific rate parameters; using a one-size-fits-all approach could result in forced
parameter differences that would not be present if the efflux rates had been specific to each
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participant. However, the lack of data describing these individualized effluxes makes this
problem difficult to address. There are also very limited studies investigating these processes
in individuals with ASD, so it is challenging to characterize any flux differences for those
participants compared to the neurotypical group. Thus, the efflux rate constants must be
assumed to be the same for every participant until more data regarding the rates of these
processes become available.
The mean values for many of the estimated parameters in the neurotypical group are close to
the biological activity rates of their corresponding enzymes that are found in the literature.
Referring to the literature values in Table 4, it can be seen that the approximate rate constant
for MAT-I + MAT-III activity is fairly close to the mean estimated value for control
participants. The estimates for the DNMT+GNMT and MS+BHMT rate constants are also
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within acceptable ranges of their literature values. Additionally, the mean estimate of the
rate constant for SAHH is very close to the true rate constant, although this is expected due
to the constraint applied during estimation. The estimated rate constants for GCL, GS, and
the GPX:GR ratio are close to their approximate nominal values as well. However, the mean
estimate for CBS is significantly different from its nominal value. It is unlikely that this
discrepancy is a consequence of the lumped homocysteine-cystathionine-cysteine reaction
used in the model. At steady state, the theoretical metabolic flux from homocysteine to
cystathionine will be equivalent to the flux from cystathionine to cysteine; since the flux

throughout the transsulfuration pathway needs to be the same at every reaction step, the
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lumped reaction rate from homocysteine to cysteine will also be equal to either of these two
individual reaction rates. Therefore, the product of the homocysteine concentration and rate
constant p5 still reflects the actual conversion rate of homocysteine to cystathionine, and p5
should be a valid approximation of CBS activity. A more likely reason for the discrepancy is
the fact that cysteine is used in many other metabolic processes besides the transsulfuration
pathway; it is also involved in the synthesis of taurine, pyruvates, sulfates, and sulfites, for
example (Stipanuk et al., 2006). The model does not describe these alternative pathways,
and thus does not account for the higher levels of cysteine that would be required to carry
out all of these reactions. This may explain why the rate of cysteine synthesis is
underestimated in the model.
Parameter estimation revealed observable differences in parameters p1, p2, p4, and p8:p9
between neurotypical and ASD participants. This suggests some degree of difference in
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activity rate for MAT-I+MAT-III, DNMT+GNMT, MS+BHMT, and GPX:GR in participants

with ASD as compared to neurotypical participants. The GPX:GR difference is expected;
the ratio of reduced to oxidized glutathione as a marker of oxidative stress has been well-
documented (Asensi et al., 1999; James et al., 2006) and there is evidence to suggest that a
decreased ratio is present in participants with ASD (Adams et al., 2011; James et al., 2006;
Melnyk et al., 2012). The DNMT+GNMT parameter represents the rate at which SAM is
converted into SAH, and reflects the SAM/SAH ratio, which is an indicator of DNA
methylation capacity (Yi et al., 2000). In participants with ASD, the SAM/SAH ratio has
been found to be significantly reduced (Adams et al., 2011; James et al., 2006; Melnyk et al.,
2012). With regards to the differences in MAT-I + MAT-III, oxidative stress has been linked
to a down-regulation of the methionine adenosyltransferase enzymes (Avila et al., 1998).
Given that increased oxidative stress is characteristic of ASD, it is possible that there could
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be altered MAT rates in participants on the spectrum. Finally, it has been found that
increased oxidative stress can be responsible for reduced MS activity (Muratore et al., 2013).
This mechanism leaves more homocysteine available to be eventually converted to
glutathione, which can then be used to lower oxidative stress. These observations are in line
with the finding that the participants with ASD had different values of the p4 parameter as
compared to their neurotypical counterparts.
Model metabolite concentrations were found to be most sensitive to changes in parameter

p6, followed by parameter p5, efflux rate constant f7, and parameter p4. These correspond to
the first-order rate constants of the enzymes GCL and CBS, the removal of GSH, and the
enzymes MS+BHMT, respectively. GCL is responsible for the conversion of cysteine to
glutamylcysteine, so the sensitivity results suggest that the formation of glutamylcysteine
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has an important role in the model. This is somewhat surprising because the formation of
GSH was expected to have a significant impact on the model predictions; instead the
synthesis of its precursor was identified as more important. That being said, the synthesis of
a GSH precursor will ultimately affect GSH formation, and formation of glutamylcysteine is
considered to be the rate-limiting step of GSH synthesis (Hamilton et al., 2003; Lu, 2009;
Shi et al., 1994). In addition, the relatively high sensitivity to the removal of GSH does
suggest that levels of GSH have a significant effect on other metabolites in the methionine
cycle and transsulfuration pathway; this could be supported by findings of redox levels

(which are influenced by GSH) affecting synthesis of certain metabolites in these pathways
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(Mosharov et al., 2000; Richman and Meister, 1975). The other important model enzymes,
CBS and MS+BHMT, are involved in the synthesis of cysteine and methionine, respectively,
both from homocysteine. Given that these enzymes form the juncture between the
methionine cycle and transsulfuration pathway, and determine the fate of homocysteine, it is
not as surprising that these are among the most important parameters in the model.
The robustness of the most important sensitivity analysis results to changes in the study
participants was verified by leave-one-out cross validation, where no substantial differences
in sensitivity measures were observed between the averaged training and test set results
(Table 6). The sensitivity results were nearly identical across training sets since each training
set’s baseline parameter values were averaged from the parameters of all neurotypical
participants except one, and removing one different participant from the average of 81
participants in each training set is unlikely to have a significant effect on the average
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parameter values. This explains why the variations in the sensitivity measures between
training sets are below the used cutoff. Unlike the training set, however, the test sets did
show some variation in their sensitivity measures for certain parameters. Despite this
difference, the test sets’ averaged results were very close to those from the averaged training
results, suggesting that the parameters’ sensitivity measures are much less dependent on
individual parameter values than on the structure of the model itself.
Model robustness is further suggested by the small values of the sensitivity measures for
each parameter. However, a potential drawback of these small sensitivities is poor
observability of the parameters in the experimental data. One possible way to address this
issue could be to implement a different measure of sensitivity, perhaps by considering a
relationship among the metabolites’ relative changes other than the Euclidean norm, or by
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replacing the relative change of a metabolite with a different calculation of change

altogether. Another solution is to employ regularization methods, such as Tikhonov
regularization (Engl et al., 1996), that can deal with poor observability by providing more
stable estimates of model parameters (Golub et al., 1999). Experimentation with Tikhonov
regularization (using neurotypical participants and values of the regularization parameter up
to 0.1) yielded parameter values that, on average, varied by less than 6% of their current
solutions. This was the case for all parameters except p8:p9, which had an average percent
change of 89.1%. These results indicate that the parameters are robust with respect to the
parameter observability, with the exception of p8:p9. However, the p8:p9 ratio is not one of
the key variables in the model as determined by sensitivity analysis, and thus the conclusions
do not change as a result of this analysis.
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Analysis of the raw plasma concentration data in (Melnyk et al., 2012) yielded many
important observations of metabolic abnormalities in study participants with ASD. Among
the primary outcomes of that study were significant measured differences for methionine,
SAM, SAH, total cysteine, GSH, and GSSG concentrations in control and case participants.
The study concluded that participants with ASD have genome-wide reduced DNA
methylation, in addition to finding evidence for increased oxidative stress in participants
with ASD. While the current model does not provide any insight into genome-wide trends, it
does offer some results similar to those obtained by analysis of the raw data. Mainly,

observed differences in the p1, p2, p4, and p8:p9 rate parameters point to equivalent
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metabolic differences identified in (Melnyk et al., 2012). An important distinction to make,

however, is that those analyses focused directly on the metabolite concentrations, while the
model analyses here are aimed toward the rates at which the metabolic reactions take place.
With this in mind, the model also produced some novel results that were not previously
extracted from the data. Sensitivity analysis found two parameters relating to the fate of
homocysteine to be very important, while no significant difference in homocysteine
concentration was found in the raw data. While this could be an important finding, it could
also be an artifact of the model; the idea of homocysteine metabolism being altered in
response to oxidative stress (Muratore et al., 2013) suggests the former, however. The
parameter pertaining to glutamylcysteine synthesis (by GCL) was indicated to be of even
greater importance, but no analysis of glutamylcysteine concentrations in the raw data was
performed. Future investigations should explore in more detail the importance of GCL, and
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its rate-limiting reaction, in GSH synthesis.
5. Conclusions
This study developed a model for two pathways of potential importance to ASD and
identified model parameters, as well as their PDFs, from clinical data from neurotypical
participants and those on the spectrum. Significant statistical differences can be seen in four
of the parameters between the two groups through comparison of their mean parameter
values and their parameter distributions. Almost all estimated rate parameters were found to
match with corresponding experimental values from the literature, with the exception of the
rate constant for cystathionine β-synthase. Sensitivity analysis revealed parameter p6, which
reflects the activity of the enzyme glutamate-cysteine ligase, to be especially important to
the model’s predictions. This finding aligns with the observation that the reaction the
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enzyme is responsible for, i.e., the formation of glutamylcysteine, is the rate-limiting step in
glutathione synthesis. Development of this model serves as the first step for identifying
targets for potential future intervention strategies. Reactions with significant differences
among the two participant groups can serve as a potential starting point for future
investigations.
Acknowledgments
The authors gratefully acknowledge partial financial support from the National Institutes of Health (Grant
R01AI110642).
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10.1074/jbc.M002725200. [PubMed: 10884384]

Highlights
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• Previous studies linked autism spectrum disorder (ASD) to metabolic

irregularities.
• A model describing transmethylation and transsulfuration pathways was

developed.
• Case-control clinical data were used to estimate distribution of model

parameters.
• Sensitivity analysis identified potential metabolic reactions of interest.
• Results of this study hint at potential intervention strategies for ASD.

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Figure 1.
The structure of the methionine cycle and transsulfuration pathway as described by the
mathematical model. Met: methionine, SAM: S-adenosylmethionine, SAH: S-
adenosylhomocysteine, H-Cys: homocysteine, Cys: cysteine, Glut-Cys: glutamylcysteine,
GSH: glutathione, GSSG: glutathione disulfide. The p variables represent participant-
specific rate parameters, while the f variables correspond to flux rate constants. Variable u is
a zero-order, participant-dependent methionine influx used to keep the system at steady
state.
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Figure 2.
PDFs of the eight participant-specific model parameters for the neurotypical and ASD
participant groups. (A) Rate parameter p1 for MAT-I + MAT-III. (B) Rate parameter p2 for
DNMT + GNMT. (C) Rate parameter p3 for SAHH. (D) Rate parameter p4 for MS + BHMT.
(E) Rate parameter p5 for CBS. (F) Rate parameter p6 for GCL. (G) Rate parameter p7 for
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GS. (H) Ratio of rate parameters p8 and p9 for GPX:GR.

Table 1
Metabolites included in and linear algebraic equations of the model.

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Metabolite Concentration Variable Model Equation

Methionine c1 0 = p4c4 − p1c1 + f2c2 + f4c4 + f7c7
S-adenosylmethionine (SAM) c2 0 = p1c1 − p2c2 − f2c2
S-adenosylhomocysteine (SAH) c3 0 = p2c2 −p3c3
Homocysteine c4 0 = p3c3 − p4c4 − p5c4 − f4c4
Cysteine c5 0 = p5c4 −p6c5
Glutamylcysteine c6 0 = p6c5 − p7c6
Glutathione (GSH) c7 0 = p7c6 − p8c7 + p9c8 − f7c7
Glutathione disulfide (GSSG) c8 0 = P8c7 − p9c8

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Table 2
Enzymes and fluxes represented by parameters used in the mathematical model.

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Parameter Representative Enzyme(s) or Flux Abbreviation

p1 methionine adenosyltransferase I MAT-I
methionine adenosyltransferase III MAT-III

p2 DNA methyltransferases DNMT
glycine N-methyltransferase GNMT

p3 S-adenosylhomocysteine hydrolase SAHH
p4 methionine synthase MS
betaine-homocysteine methyltransferase BHMT

p5 cystathionine β-synthase CBS
p6 glutamate-cysteine ligase GCL
p7 glutathione synthetase GS
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p8 glutathione peroxidase GPX
p9 glutathione reductase GR
f2 SAM efflux –
f4 homocysteine efflux –
f7 GSH efflux –
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Table 3
Means and standard deviations of metabolite concentrations for the neurotypical and ASD groups.
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Concentration Mean ± SD (μM)
Metabolite Neurotypical (n = 82) ASD (n = 93) p-value

Methionine 23.1 ± 3.6 19.0 ± 2.9 < 0.0001
SAM 0.0696 ± 0.0100 0.0600 ± 0.0104 < 0.0001
SAH 0.0152 ± 0.0036 0.0186 ± 0.0047 < 0.0001
Homocysteine 4.74 ± 1.01 5.10 ± 1.20 0.0323
Cysteine 209 ± 17 190. ± 18 < 0.0001
Glutamylcysteine 2.33 ± 0.59 1.85 ± 0.48 < 0.0001
GSH 2.31 ± 0.70 1.71 ± 0.36 < 0.0001
GSSG 0.147 ± 0.058 0.236 ± 0.100 < 0.0001
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Table 4
Estimates of control parameters when p1 through p4 are individually constrained. All parameters have units of hr−1 except for p8:p9, which is
dimensionless.
Vargason et al.
Mean Parameter Value ± SD Literature value
Parameter p1 p2 P3 P4 Reference
constrained constrained constrained constrained
p1 4.90 ± 0.78 0.412 ± 0.064 1.90 ± 0.30 2.97 ± 0.46 4.78 (Broxson et al., 1989; del Pino et al., 2000; Nijhout et al., 2006; Sullivan and
Hoffman, 1983)
p2 1610 ± 218 127 ± 17 620. ± 84 976 ± 140. 125 (Castro et al., 2003; Flynn and Reich, 1998; Luka and Wagner, 2003; Nijhout et al.,
2006)
p3 7720 ± 2215 610. ± 175 2970 ± 852 4680 ± 1410 2780 (Castro et al., 2003; Døskeland and Ueland, 1982; Nakanishi et al., 2001)
p4 22.3 ± 4.6 −0.0210 ± 0.5858 7.38 ± 1.62 12.7 ± 2.6 12.2 (Castro et al., 2003; Finkelstein and Martin, 1984)
p5 1.93 ± 0.70 1.93 ± 0.70 1.93 ± 0.70 1.93 ± 0.70 10.4 (Castro et al., 2003; Finkelstein and Martin, 1984)
p6 0.0421 ± 0.0125 0.0421 ± 0.0125 0.0421 ± 0.0125 0.0421 ± 0.0125 0.0276 (Davis et al., 2006; Huang et al., 1988; Toroser et al., 2006)
p7 3.82 ± 0.77 3.82 ± 0.77 3.82 ± 0.77 3.82 ± 0.77 10.0 (Lyons et al., 2000; Martensson, 1987)
p8:p9 0.0660 ± 0.0244 0.0660 ± 0.0244 0.0660 ± 0.0244 0.0660 ± 0.0244 0.0118 (Blanco et al., 2007; López-Barea and Lee, 1979; Maddipati and Marnett, 1987)

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Table 5
Comparison of model parameter estimates for control and ASD participants. All parameters have units of hr−1
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except for p8:p9, which is dimensionless.
Parameter Mean ± SD
Parameter Neurotypical (n = 82) ASD (n = 93) p-value

p1 1.90 ± 0.30 2.81 ± 0.42 < 0.0001
p2 620. ± 84 889 ± 154 < 0.0001
p3 2970 ± 852 3020 ± 1020 0.726
p4 7.38 ± 1.62 9.32 ± 2.00 < 0.0001
p5 1.93 ± 0.70 1.35 ± 0.47 < 0.0001
p6 0.0421 ± 0.0125 0.0346 ± 0.0088 < 0.0001
p7 3.82 ± 0.77 3.59 ± 0.63 0.031

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p8:p9 0.0660 ± 0.0244 0.144 ± 0.065 < 0.0001

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Table 6
Results of sensitivity analysis from perturbing each parameter and efflux rate constant by 1%. Also shown are
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the results of leave-one-out cross validation for this analysis.
Parameter or Efflux Rate Constant Sensitivity (all samples) Mean Training Sensitivity ± SD Mean Test Sensitivity ± SD
p1 0.0099 0.0099 ± 0.0000 0.0099 ± 0.0000
p2 0.0100 0.0100 ± 0.0000 0.0100 ± 0.0000
p3 0.0100 0.0100 ± 0.0000 0.0100 ± 0.0000
p4 0.0136 0.0136 ± 0.0000 0.0136 ± 0.0011
p5 0.0168 0.0168 ± 0.0000 0.0168 ± 0.0008
p6 0.0261 0.0261 ± 0.0000 0.0261 ± 0.0001
p7 0.0100 0.0100 ± 0.0000 0.0100 ± 0.0000
p8: p9 0.0100 0.0100 ± 0.0000 0.0100 ± 0.0000
f2 0.0001 0.0001 ± 0.0000 0.0001 ± 0.0000

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f4 0.0000 0.0000 ± 0.0000 0.0000 ± 0.0000
f7 0.0141 0.0141 ± 0.0000 0.0141 ± 0.0000

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Published in final edited form as:

Mathematical Modeling of the Methionine Cycle and

Autism spectrum disorder (ASD) is a general diagnosis for a group of neurodevelopmental

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

detoxification of environmental toxins, including heavy metals (Adams et al., 2011).

2. Materials and Methods

oxo-deoxyguanosine in DNA were also quantified using a HPLC-ultraviolet system and

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

2.2. Model development

A compartmental model, where the concentrations of the metabolites within each

transsulfuration pathway. Cystathionine, an intermediate in the reaction of homocysteine

The model consists of 8 equations (Table 1) and contains 9 participant-specific parameters

participant’s three efflux rates to achieve a steady state balance:

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

corresponds to the combined activities of methionine adenosyltransferase I (MAT-I) and

reductase (GR). These model enzymes are summarized in Table 2.

2.3. Parameter estimation

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

2.4. Model assumptions

pathway behavior reasonably well.

3. Changes in concentrations of metabolites in plasma are reflected by proportional

4 The conversion of methionine to SAM is due to the combined effects of MAT-I

5 The conversion of SAM to SAH is due to the combined contributions of DNMT

6 While the reaction converting SAH to homocysteine by the enzyme SAHH is

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

7 The conversion of homocysteine to methionine is due to the combined

contributions of MS and BHMT. This assumption is made for reasons similar to

8 The reactions involving the conversion of homocysteine to cystathionine and the

9 There are no indirect inhibitory or regulatory effects between metabolites in the

2.5. Sensitivity analysis

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

considered in this cross-validation.

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

3.3. Sensitivity analysis

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

had different metabolite concentrations, resulting in different model parameters.

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

activity rate for MAT-I+MAT-III, DNMT+GNMT, MS+BHMT, and GPX:GR in participants

Model metabolite concentrations were found to be most sensitive to changes in parameter

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

replacing the relative change of a metabolite with a different calculation of change

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

metabolic differences identified in (Melnyk et al., 2012). An important distinction to make,

its rate-limiting reaction, in GSH synthesis.

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

Validation. PLoS ONE. 2017; 12(1):e0169526.doi: 10.1371/journal.pone.0169526 [PubMed:

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

10.1021/bi9810609. [PubMed: 9790680]

https://doi.org/10.3346/jkms.2005.20.5.721. [PubMed: 16224142]

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

determination of oxidized and reduced plasma aminothiols using coulometric electrochemical

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

Rai V. Association of methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism with

doi.org/10.1007/s11011-016-9815-0. [PubMed: 26956130]

a US metropolitan area. Journal of the American Medical Association. 2003; 289(1):49–55.

J Theor Biol. Author manuscript; available in PMC 2018 March 07.

• Previous studies linked autism spectrum disorder (ASD) to metabolic

• A model describing transmethylation and transsulfuration pathways was

• Case-control clinical data were used to estimate distribution of model

• Sensitivity analysis identified potential metabolic reactions of interest.

• Results of this study hint at potential intervention strategies for ASD.